Difference between revisions of "Koch Lab:Protocols/Microsphere-DNA tethering"

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(New page: {{Koch_Lab}} <div style="padding: 10px; width: 720px; border: 5px solid #008;"> ==Principle== ==Some general thoughts--not rigorously tested== ==A specific protocol, 4411 basepair PCR-labe...)
 
 
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==Principle==
 
==Principle==
==Some general thoughts--not rigorously tested==
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[[Image:Glass-DNA-Microsphere Tether.png|right|thumb|Schematic of microsphere-DNA tethering]]
==A specific protocol, 4411 basepair PCR-labeled DNA, 0.5 micron streptavidin microspheres==
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A microsphere can be tethered to a microscope slide (or other surface) via a single double-stranded DNA molecule (dsDNA).  Commonly, the microsphere is about a 500 nm polystyrene or silica microsphere, but of course many other possibilities exist, such as: 2.8 micron polystyrene microspheres with embedded maghemite superparamagnetic nanocrystals; small gold nanoparticles; quantum dots.  Once tethers have been formed, they can be stretched with optical tweezers (e.g., to measure the elastic properties of DNA, or unzip the DNA), or the DNA tether length can be monitored via analysis of the Brownian motion of the microsphere (Tethered Particle Motion, TPM...perhaps to detect DNA looping by a protein).
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To achieve tethering, you need to attach one end of the DNA to the glass slide, and the other end to the microsphere.  Generically, this is what needs to be done:
 +
glass --- connection#1 --- endDNA******DNAend --- connection#2 --- microsphere
 +
 
 +
A very common method that is used is to label the DNA with [http://en.wikipedia.org/wiki/Digoxigenin digoxigenin (dig)] on one end and [http://en.wikipedia.org/wiki/Biotin biotin] on the other end.  The surface of the glass slide is coated with a polyclonal antibody against dig (anti-dig) and the microspheres are coated with streptavidin (which binds to biotin with high affinity).  This makes the tethering scheme:
 +
glass --- anti-dig --- digDNA******DNAbiotin --- streptavidin --- microsphere
 +
 
 +
There are also many other methods.  Because of the availability of a variety of streptavidin coated microspheres, biotin-streptavidin connection seems to be by far the most popular.  Dig/anti-dig is also very common, but other methods for tethering that end of the dNA to the class are:
 +
*anti-HA --- HA-RNA polymerase-DNA
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*gold --- thiol-DNA
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*anti-his --- his-Protein-DNA
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*anti-fluorescein --- fluoresceinDNA
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==Specific protocols==
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*[[/Glass, dig, biotin, microsphere, 4kb DNA|4411 basepair PCR-labeled DNA (dig, biotin), 0.5 micron streptavidin microspheres]]
  
 
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[[Category:Protocol]] [[Category:DNA]]  
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[[Category:Protocol]] [[Category:DNA]] [[Category:Microscopy]]
 
[[Category:Koch Lab Page]]
 
[[Category:Koch Lab Page]]

Latest revision as of 22:36, 30 October 2007

Moving pan.gif

Principle

Schematic of microsphere-DNA tethering

A microsphere can be tethered to a microscope slide (or other surface) via a single double-stranded DNA molecule (dsDNA). Commonly, the microsphere is about a 500 nm polystyrene or silica microsphere, but of course many other possibilities exist, such as: 2.8 micron polystyrene microspheres with embedded maghemite superparamagnetic nanocrystals; small gold nanoparticles; quantum dots. Once tethers have been formed, they can be stretched with optical tweezers (e.g., to measure the elastic properties of DNA, or unzip the DNA), or the DNA tether length can be monitored via analysis of the Brownian motion of the microsphere (Tethered Particle Motion, TPM...perhaps to detect DNA looping by a protein).

To achieve tethering, you need to attach one end of the DNA to the glass slide, and the other end to the microsphere. Generically, this is what needs to be done:

glass --- connection#1 --- endDNA******DNAend --- connection#2 --- microsphere

A very common method that is used is to label the DNA with digoxigenin (dig) on one end and biotin on the other end. The surface of the glass slide is coated with a polyclonal antibody against dig (anti-dig) and the microspheres are coated with streptavidin (which binds to biotin with high affinity). This makes the tethering scheme:

glass --- anti-dig --- digDNA******DNAbiotin --- streptavidin --- microsphere

There are also many other methods. Because of the availability of a variety of streptavidin coated microspheres, biotin-streptavidin connection seems to be by far the most popular. Dig/anti-dig is also very common, but other methods for tethering that end of the dNA to the class are:

  • anti-HA --- HA-RNA polymerase-DNA
  • gold --- thiol-DNA
  • anti-his --- his-Protein-DNA
  • anti-fluorescein --- fluoresceinDNA

Specific protocols